1. Field of the Invention
The present invention relates to plant choline monooxygenase which is involved in the occurence of plant injury attributable to dry or saline soil, which is believed to contribute to the improve of plant tolerance to dry or saline soil, and which is induced by dry or saline soil, as well as a gene encoding the choline monooxygenae.
2. Descriptionof the Related Art
At presnt, desertification or salt accumulation in cultivated land is progressing in a large is number of areas on the earth. These envinmental changes considered as serious issues in connection with current envirormental problems and food problems in the 21st century. As means to solve these problems, the breeding of environmental stress-resistant plants is attracting attentiou together with engineering-based solutions as irrigation.
Specific examples of damage caused by salt acmulafn may be enumerated as follows: (1) because of accumulated salts in soil, moisture potential in soil decems, which makes it impossibe for plants to absorb moisture; (2) because of salts which have been absorbed (or which have invaded) into plant bodies plant metabolism is disturbed; and (3) becuse of accumulated salts, absorption of other ions necessary for plant growth is inhibited (Fumihiko Sato, Plant Cell Engineering, an extra issue, xe2x80x9cEnvirnmental Problems and Bioologyxe2x80x9d, pp. 33-39, 1994). In particular, inhibition of moisture absorption caused by dehydration, salt injury, etc. eventually decreases photosynthesis activity to thereby inhibit the growth of plants
The existence of adaptation mianisms against stresses such as dehydration or salts has become evident in microorganisms and plants. Among all, compatible solutes (i.e. low molecular weight orgaic compounds or osmoregulating substances) have been investigated vigorously. Compatible solutes are those substances which are characterized by having low molecular weights, being rich in water-solubility and difficult to metbolize, and not affecting metabolismn. As specific examples of compatible solutes, amphoteric compounds such as glycine betaine, proline, and polyols such as pinitol, sorbitol, mannitol are known. In particular, glycine betain (hereinafter, referred to as xe2x80x9cbetainexe2x80x9d) is utlized widely not only in higher plants such as chenopodiaccous plants gramineous plants and solanaceous plants, but also in microorganism. It is noted that this compatible solute is functioning in protecting proteins from high temperature stress (Paleg, L. G., et al., Aust. J. Plant Physiol. 8:107-114, 1981; Allakhdverdiev S. I., J. Photochem. Pbotoiol. 34:149157, 1996), in maintaining osmotic pressure balance against the environment (Robinson, S. P. and Jones, G. P., Aust. J. Plant Physiol. 13:659-668, 1986) and in protecting soluble enzyes from salt stress (Gabbay-Azaria et al., Arch. Biochem Biophys. 264:333-339, 1988).
In spinsh which is well studied among higher planis, betaine is syn in two steps through choline and betaine aldehyde. Specifically oxidization in the first step is catalyzed by a ferredoxin-dependent choline monooxygenase (Brouquisse, R. et al., Plant Physiol. 90:322-329, 1989), and oxidization in the second step is catalyzed by a NAD-dependent betaine aldehyde dehydrogenase (Weretilnyk, E. A. et al., Plamta. 178: 342-352, 1999). It is confirmed that when such aplant is exposed to salt stress, the activity of each of the above enzymes rises and the amount of betaine is increased (Hanson, A. D. et al., Proc. Natl. Acid. Sci, U.S.A. 82: 3678-3682, 1985).
A choline oxidase obtained from a Gram-negative soil bacterium, Arthrobacter globtformis is able to oxidize choline to betaine in one step oxidiaon (Ikuta S. et al., J. Biochem. 82: 1741-1749, 1977).
Several attempts have been made to accunulate betaine in plants and confer salt tolemace on them by incorporating in plant bodies two enzyme genes from Escherichia coli and a higher plant or a choline oxidase gene and allowing the gene constant expression. Accumuation of betaine in plant bodies have been reported when Arthrobacter globiformis codA gene (WO96/29857), E. coli beta gene (Japanese Unexamined Patent Publicaton No. 10-191983) and spinach CMO gene (Nuccio, M. L. et al., The Plant J., 16: 487-496, 1998) weme incorporated However, no attempts have succeeded in accumulating betaine at such levels as seen in salt tolent plants. Thus, it is desired to establish a betaine synthesis system which can accumulate betaine in plants at high levels.
It is the object of the invention to provide a choline monooxygenase, a gene encoding the same, a vector comprising the gene, a transformmt comprising the vector, a stress resistant plan and a method for inducing betaine accumulation.
As a result of intensive and extensive researches toward the solution of the above problem, the present inventors have isolated a full-length choline monooxygenae gene inducible by dry and saline soil from Chenopodium album L. which exhibits tolerance to dry and saline soil and can accumulate betaine at a high rate of about 60 xcexcmol/g fresh weight under salt stress, and found that this gene is capable of acumulating betaine in plant bodies. Also, the inventors have found for the first time that a transit peptide sequence of the choline monooxygenase gene induces protein accumulation umder salt stress. Thus, the present invention has been achieved.
The present invention relates to the following reombinant protein (a) or (b):
(a) a protein comprising the amino acid sequence shown in SEQ ID NO: 2, 4 or 6;
(b) a protein which comprises the amino acid sequence shown in SEQ ID NO: 2, 4 or 6 having deletion, substitution or addition of one or several amino acids, and which has choline monooxygenase activity.
The present invention further relates to a choline monooxygenase gene encoding the above described protein.
The present invention further relates to a gene comprising the following DNA (c) or (d)
(c) a DNA comprising the nucleotide sequence shown in SEQ ID NO: 1, 3 or 5;
(d) a DNA which hybridizes to a DNA cormprising the nuclcotide sequence shown in SEQ ID NO: 1, 3 or 5 under stringent conditions and which encodes a protein having choline monooxygenase activity.
The present invention fuiwr relates to a recombinant vector comprising the above described gene.
The present invention further relates to a recombinant comprising the above-described recombinant vector.
The present invention further relates to a method for producing a choline monooxygenae comprising culturing the above-described transformant and recovering the choline monooxygenase from the resultant culture.
The present invention further relates to the following peptide (e) or (f):
(e) a peptide comprismg the amino adid sequence shown in SEQ ID NO: 17;
(f) a peptide which comprises the amino acid sequence shown in SEQ ID NO: 17 having deletion, substitution or addition of one or several amino acids and which has signal peptide activity; or a salt thereof.
The present invention further relates to a gene encoding the above-described peptide. Specific examples of the gene include a gene comprising the foUowing DNA (g) or (h):
(g) a DNA comsing thw nucleotide sequenc shown in SEQ ID NO: 16;
(h) a DNA which hybridizes to a DNA comprising the nucleotide sequence shown in SEQ ID NO: 16 under stringent conditions and which encodes a protein having signal peptide activity.
The present invention fisrher relates to a recombinant vector comprising a gene encoding the above-described peptide and a gene of interest. As the gene of interest, a gene which leads to production of a polypeptide or production of a plant metabolite (e.g. a substance that confers stress resistance), or Chenopodium album choline monooxygenase gene may be enumerated.
The present invention further relates to a transformant comprising the recombinant vector comprising a gene encoding the above-discribed peptide and a gene of intrest. Specific examples of the transformant include a plant body, plant organ, plant tissue and cultured plant cell.
The present invention further relates to a method for creating an enviernmental stress-resistant plant, comprising culturing or cultivating a transformed plant comprising the above-described recombinant vector under environmental stress (e.g. salt stress) conditions; or an environmental stress-resistant plant created by this method.
The present invention further relates to a method for inducing accumulation of a polypeptide or a plant metabolite (e.g. a subslance that confers envronmental stress resistance), or cultivating the above-described transfromant under environmental stress conditions. As a specific example of the substance that confers environmental stres reistance, betaine may be given.
The present invention further relates to a method for producing betaine, comprising culturing or cultivating a transformant comprising the recombinant vector comprising a gene econcoding the above-described peptide and a choline nonooxygenase gene and then recovering betaine from the resultant culture or the cultivated product.
This speccation includes part or all of the contents as disclosed in the specification and or drawings of Japanese Patent Application No.11-273275, which is a priority document of the present application.